Izumi Tanaka

Microarray-based global mapping of integration sites for the retrotransposon, intracisternal A-particle, in the mouse genome.

Mammalian genomes contain numerous evolutionary harbored mobile elements, a part of which are still active and may cause genomic instability. Their movement and positional diversity occasionally result in phenotypic changes and variation by causing altered expression or disruption of neighboring host genes. Here, we describe a novel microarray-based method by which dispersed genomic locations of a type of retrotransposon in a mammalian genome can be identified. Using this method, we mapped the DNA elements for a mouse retrotransposon, intracisternal A-particle (IAP), within genomes of C3H/He and C57BL/6J inbred mouse strains; consequently we detected hundreds of probable IAP cDNA–integrated genomic regions, in which a considerable number of strain-specific putative insertions were included. In addition, by comparing genomic DNAs from radiation-induced myeloid leukemia cells and its reference normal tissue, we detected three genomic regions around which an IAP element was integrated. These results demonstrate the first successful genome-wide mapping of a retrotransposon type in a mammalian genome.

Unusual long target duplication by insertion of intracisternal A-particle element in radiation-induced acute myeloid leukemia cells in mouse

Retrotransmission into the IL‐3/GM‐CSF gene locus by the retrotransposon intracisternal A‐particle (IAP) had been observed in distinct tumor cell lines. We analyzed the locus in genomes from 7 different myeloid leukemia cell strains which were originally generated by whole‐body X‐irradiation of the inbred C3H/He mice at a dose of 3 Gy and maintained by in vivo passage. In one leukemia cell strain out of 7 such cases, RFLP of an allele of the interleukin‐3 gene was found. Sequence analysis after cloning from the genomic library showed that a type IΔ2 IAP element was inserted in the region upstream of the IL‐3 gene in the head‐to‐head orientation. This suggests that the locus in myeloid cells is sensitive for integration of IAP elements. Additionally, an unusual long target duplication of 82 bp, 14‐fold larger than normal one, was found at the junction of the element. This suggests the possibility of a radiation‐induced integration mechanism which is distinct from normal retrotransmission.

Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation.

Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome-cell interactions are crucial, but they are not well understood. Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.

Acceleration of Regeneration of Mucosa in Small Intestine Damaged by Ionizing Radiation Using Anabolic Steroids

Abstract Damage to intestine is a serious problem after accidental radiation exposure. To examine substances to ameliorate damage by postirradiation administration, we focused on the regeneration process after irradiation of the intestine. Using experimental systems, the effects of clinically used sex hormones on regeneration were compared. An anabolic steroid, nandrolone (19-nortestosterone), stimulated proliferation in IEC-6 epithelial cells. A single injection of 19-nortestosterone ester with prolonged action into mice 24 h after abdominal irradiation at a lethal dose of 15.7 Gy showed significant life-saving effects. Regeneration indicators such as microcolonies of BrdU-incorporated cells at day 5 and c-myb mRNA expression levels at day 4 were enhanced by 19-nortestosterone administration. In contrast, high concentrations of estradiol inhibited growth of IEC-6 cells. Treatment of abdominally irradiated mice with estradiol ester decreased levels of regeneration indicators and survival. These results suggest the effectiveness of the anabolic steroid as well as the importance of manipulation of steroid receptors in the recovery of mucosa damaged by radiation.

Detection and cloning of unique integration sites of retrotransposon, intracisternal A-particle element in the genome of acute myeloid leukemia cells in mice

We previously found retrotransposition of the intracisternal A‐particle (IAP) element in the genome of acute myeloid leukemia (AML) cells induced by X‐irradiation of C3H/He mice (FEBS 16333). To analyze the occurrence of the IAP‐mediated retrotransposition in AML cells, we compared integration sites of the IAP element by polymerase chain reaction (PCR) in the genomes of five AML strains derived from different C3H mice. Unique PCR products were found in all of the above independent leukemia cells, whereas no such products were detected in normal cells. Results of cloning, sequencing and Southern analyses showed that the PCR products were derived from novel integration sites of the IAP element in the genome. The data suggest that IAP‐mediated retrotransposition occurs frequently in radiation‐induced AML cells from C3H/He mice.

Increased expression of intracisternal A-particle RNA in regenerated myeloid cells after X irradiation in C3H/He inbred mice.

Abstract Ishihara, H., Tanaka, I., Furuse, M. and Tsuneoka, K. Increased Expression of Intracisternal A-Particle RNA in Regenerated Myeloid Cells after X Irradiation in C3H/He Inbred Mice. Myeloid leukemia cells were derived from regenerated hematopoietic cells damaged by sublethal doses of X radiation in C3H/He inbred mice. We previously found that within the genome of the myeloid leukemia cells, a retrotransposon, the intracisternal A-particle (IAP) element, is integrated. Levels of IAP RNA, the source of cDNA for the integration, were analyzed quantitatively in C3H mice. Higher levels of IAP transcripts were observed in normal cells, particularly in hematopoietic cells, from C3H/He mice, than in those from C57BL/6J and STS/A mice. In the C3H/He mice, an approximately twofold increase in IAP RNA was found in the regenerated spleen and bone marrow cells at 5 days and from 12 to 90 days after whole-body X irradiation. In addition, an increased level of IAP RNA was observed in all the myeloid leukemia cells derived from C3H/He mice. This suggests that the elevated levels of IAP RNA in the regenerated hematopoietic cells after irradiation contribute to the increase in retrotransposition of IAP found in myeloid leukemia cells from C3H/He mice.

Comparison of Absorbents and Drugs for Internal Decorporation of Radiocesium: Advances of Polyvinyl Alcohol Hydrogel Microsphere Preparations Containing Magnetite and Prussian Blue.

Radiocesium nuclides, used as a gamma ray source in various types of industrial equipments and found in nuclear waste, are strictly controlled to avoid their leakage into the environment. When large amounts of radiocesium are accidentally incorporated into the human body, decorporation therapy should be considered. Although standard decorporation methods have been studied since the 1960s and were established in the 1970s with the drug Radiogardase(®) (a Prussian blue preparation), application of recent advances in pharmacokinetics and ethical standards could improve these methods. Here we designed a modern dosage form of hydrogel containing cesium-absorbents to alleviate intestinal mucosa irritation due to the cesium-binding capacity of the absorbents. The effectiveness of the dosage form on fecal excretion was confirmed by quantitative mouse experiments. The total cesium excretion rate of the crystal form (1.37±0.09) was improved by the hydrogel form (1.52±0.10) at the same dose of Prussian blue, with a longer gastrointestinal tract transit time. Using a mouse model, we compared the effects of several drugs on fecal and urinary excretion of internal cesium, without the use of absorbents. Only phenylephrine hydrochloride significantly enhanced cesium excretion (excretion rate of 1.17±0.08) via the urinary pathway, whereas none of the diuretic drugs tested had this effect. These findings indicate that modifying the dosage form of cesium absorbents is important for the decorporation of internal radiocesium contamination.

Pharmaceutical drugs supporting regeneration of small-intestinal mucosa severely damaged by ionizing radiation in mice

Accidental exposure of the abdomen to high-dose radiation leads to severe consequences initiated by disruption of the mucosa in the small intestine. Therapeutic options are limited, even though various treatments have been investigated, particularly in the field of regenerative therapy. In order to identify readily available treatment methods, we included several current pharmaceutical drugs, for which the clinical trials have already been completed, in tests on mice that had undergone severe mucosal damage by radiation. The drugs were injected into mice 24 h after exposure to 15.7 Gy X-rays. The effects of the drugs on the damaged mucosa of the small intestine were evaluated using early regeneration indices [the expression of c-myb mRNA, and proliferation of epithelial cells in the form of microcolonies (MCs) by Days 4 and 5 post-irradiation] and the survival rate of the mice. Enhancement of mucosal regeneration at Day 4 (c-myb: P < 0.01, MC: P < 0.05) and improvement of the survival rate (P < 0.05) were observed when a clinical dose of gonadotropin, a stimulator of androgen, was injected. Similarly, a clinical dose of thiamazole (which prevents secretion of thyroid hormone) stimulated mucosal growth by Day 5 (c-myb: P < 0.01, MC: P < 0.05) and also improved the survival rate (P < 0.05). The nonclinical drugs histamine and high-dose octreotide (a growth hormone antagonist) also gave significant survival-enhancing benefits (P < 0.01 and P < 0.05, respectively). These results can be used to construct therapeutic programs and applied in various experimental studies to control the regeneration of damaged mucosa.

Induction of heme oxygenase-1 by whisky congeners in human endothelial cells.

It is expected that the production of the cytoprotective heme oxygenase-1 (HO-1) protein in endothelial cells would reduce severity of vascular injuries, while phenolic compounds are known to induce HO-1 mRNA and protein in various cells. We investigated the activation of HO-1 by whisky, which contains various phenolic substances. The congeners of whisky stored from 4 to 18 y in oak barrels were shown to induce an increase of HO-1 protein in human umbilical vein endothelial cells, while those of freshly distilled whisky spirit exhibited no activity. To determine the compounds with potent HO-1-inducing activity among the whisky congeners, several chemicals that had been reported to exist in whisky or oak barrels were screened, and coniferyl aldehyde and sinapyl aldehyde showed the activity. Thus, compounds that emerged in whisky during barrel storage induced cytoprotective protein, HO-1, in human endothelial cells.

Quantification of damage due to low-dose radiation exposure in mice: construction and application of a biodosimetric model using mRNA indicators in circulating white blood cells

Abstract Biodosimetry, the measurement of radiation damage in a biologic sample, is a reliable tool for increasing the accuracy of dose estimation. Although established chromosome analyses are suitable for estimating the absorbed dose after high-dose irradiation, biodosimetric methodology to measure damage following low-dose exposure is underdeveloped. RNA analysis of circulating blood containing radiation-sensitive cells is a candidate biodosimetry method. Here we quantified RNA from a small amount of blood isolated from mice following low-dose body irradiation (<0.5 Gy) aimed at developing biodosimetric tools for situations that are difficult to study in humans. By focusing on radiation-sensitive undifferentiated cells in the blood based on Myc RNA expression, we quantified the relative levels of RNA for DNA damage-induced (DDI) genes, such as Bax , Bbc3 and Cdkn1a . The RNA ratios of DDI genes/ Myc in the blood increased in a dose-dependent manner 4 h after whole-body irradiation at doses ranging from 0.1 to 0.5 Gy (air-kerma) of X-rays, regardless of whether the mice were in an active or resting state. The RNA ratios were significantly increased after 0.014 Gy (air-kerma) of single X-ray irradiation. The RNA ratios were directly proportional to the absorbed doses in water ranging from 0.1 to 0.5 Gy, based on gamma-irradiation from 137 Cs. Four hours after continuous irradiation with gamma-rays or by internal contamination with a beta-emitter, the increased RNA ratios resembled those following single irradiation. These findings indicate that the RNA status can be utilized as a biodosimetric tool to estimate low-dose radiation when focusing on undifferentiated cells in blood.